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Results for “Multiplex PCR”

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At least 379 records · Page 21Linked to original sources

PubMed2003

Identification of fast and slow growing rhizobia nodulating soybean (Glycine max [L.] Merr) by a multiplex PCR reaction.

Two DNA fragments, a 730-bp and a 900-bp fragment, one homologous to host cultivar specificity genes nolBT of Sinorhizobium fredii and the other one homologous to RSalpha, an insertion-like sequence present in Bradyrhizobium japonicum, were generated by polymerase chain reaction (PCR) with two pairs of primers. The amount of each fragment generated by the multiplex PCR was proportional to the amount of template DNA present. The amplification of the 900-bp RSalpha fragment was more sensitive, since it was amplified from a smaller amount of template DNA than the 730-bp nolBT fragment. By running the multiplex reaction in the presence of template DNA isolated from different sources, we confirmed that the reaction can discriminate between S. fredii, Bradyrhizobium japonicum and Sinorhizobium xinjiangensis.

Bradyrhizobium↗

PubMed2006

Cloning of the mating type locus from Ascochyta lentis (teleomorph: Didymella lentis) and development of a multiplex PCR mating assay for Ascochyta species.

The mating type (MAT) locus of the lentil pathogen, Ascochyta lentis, was cloned and characterized using thermal asymmetric interlaced and inverse PCR with primers designed to the HMG-box of Ascochyta rabiei. A multiplex PCR assay for mating type was developed based on MAT idiomorph and flanking sequences. Primers were designed to specifically amplify MAT from several Ascochyta spp. including A. pisi, A. fabae and A. viciae-villosae in addition to A. lentis. Four hundred and fifty and 700 bp fragments were amplified from MAT1-1 and MAT1-2 isolates, respectively, and fragment size correlated perfectly with laboratory crosses using mating type tester strains. MAT-specific PCR allowed rapid scoring of mating type in crude DNA extracts from geographically diverse population samples of A. viciae-villosae from California and Washington State, USA. This co-dominant MAT-specific PCR assay will be a valuable tool for studying the population structure, biology and epidemiology of these fungi.

Amino Acid Sequence↗

PubMed2004

Dentification of four vectors of human Plasmodium spp. by multiplex PCR: Anopheles rangeli, An. strodei, An. triannulatus, and An. trinkae (Diptera: Culicidae: Nyssorhynchus).

One of the major obstacles for studies of the biology, ecology, and behavior of Neotropical vectors of human Plasmodium has been the lack of reliable and efficient means of identifying many species. Although the subgenus Nyssorhynchus includes most species responsible for human transmission in South America, there are no polymerase chain reaction (PCR)-based techniques for identifying members of this taxon. We describe the first multiplex PCR for identifying four species in the subgenus Nyssorhynchus that are vectors of human Plasmodium spp. Four species specific primers, together with a universal primer that anneals to the 5.8S rDNA region, produce amplicons of the internal transcribed spacer two with base pair sizes of 131,308,371, and 441 for An. triannulatus, An. trinkae, An. strodei, and An. rangeli, respectively.

Animals↗

PubMed2002

Constructing universal multiplex PCR systems for comparative genotyping.

Analysis of length polymorphisms at STR loci in the human genome has become a standard approach for comparative genotyping in many areas including disease research and diagnostics, parentage assessment, investigations of human diversity, and forensic science. The simultaneous analysis of multiple STR loci through multiplex PCR and multicolor fluorescence detection offers sample conservation, high throughput, and automated genetic analysis. Careful design and optimization of tetranucleotide STR multiplexes has led to reliable, standardized systems that powerfully differentiate and distinguish individual human DNA profiles. The development of these multiplex systems involved a rigorous experimental strategy that included careful selection of PCR primer sequences (for yield, specificity, and multiplex compatability), along with optimization of PCR component concentrations, thermal cycling parameters, and fluorescence detection conditions. This developmental approach rendered well-characterized DNA typing systems that are high performing (sensitive, specific, and balanced), optimized to universal parameters (same reaction conditions), resilient to fluctuations in reaction conditions, and simple to implement and use routinely.

DNA↗

PubMed1997

Multiplex PCR for simultaneous detection of the most frequent T cell receptor-delta gene rearrangements in childhood ALL.

A rapid and simple multiplex polymerase chain reaction (PCR) is described that is capable of identifying the six most frequent rearrangements of the T cell receptor (TCR)-delta gene segments in childhood acute lymphoblastic leukemia (ALL). The PCR products amplified in a single reaction are of different size for each TCR-delta gene rearrangement. Therefore, they are readily and unambiguously distinguished after agarose gel electrophoresis and assigned to a specific V-D-J gene rearrangement. There is no need for labor-intensive and time-consuming Southern blot hybridization or nested PCR. To evaluate the multiplex assay we chose 45 DNA samples of childhood ALL analyzed beforehand for TCR-delta gene rearrangements by Southern blot and single PCR of which 30 showed TCR-delta gene rearrangements. The multiplex PCR results corresponded to the Southern blot and single PCR analyses. The described multiplex PCR enables the detection of clonal markers in about 50% of patients in order to monitor minimal residual disease (MRD) in prospective studies with a high turnover of samples.

Blotting, Southern↗

PubMed1995

Simultaneous detection by consensus multiplex PCR of high- and low-risk and other types of human papilloma virus in clinical samples.

A consensus multiplex PCR (CM-PCR) technique was developed to detect high-risk (HPV 16/18), low-risk (HPV 6/11), and over 40 other types of human papillomavirus (HPV), separately but simultaneously, by mixing three pairs of consensus primers in the same PCR mixture, for gene amplification. Simultaneous detection of three groups of HPV DNA provides valuable information for clinical practice and this procedure is simple and convenient for routine laboratory examinations. We detected HPV DNA sequences in plasmid HPV DNA and DNA extracted from tissues of condyloma acuminata and cervical carcinoma and from exfoliated cells of the lower genital tract of healthy Chinese women living in the People's Republic of China. We confirmed that this simple, convenient, and cost-beneficial CM-PCR technique is reliable for the detection of HPV DNA sequences.

Adolescent↗

PubMed2005

Rapid differentiation of Mycobacterium bovis and Mycobacterium tuberculosis based on a 12.7-kb fragment by a single tube multiplex-PCR.

The aim of this work was the design and validation of a rapid and easy single tube multiplex-PCR (m-PCR) assay for the unequivocal differential detection of Mycobacterium bovis and Mycobacterium tuberculosis. Oligonucleotide primers were based on the uninterrupted 229-bp sequence in the M. bovis genome and a unique 12.7-kb insertion sequence from the M. tuberculosis genome, which is responsible for species-specific genomic polymorphism between these two closely related pathogens. The m-PCR assay was optimized and validated using 22 M. bovis and 36 M. tuberculosis clinical strains isolated from diverse host species and 9 other non-tuberculous mycobacterial (NTM) strains. The designed primers invariably amplified a unique 168-bp (M. bovis-specific) and 337-bp (M. tuberculosis-specific) amplicon from M. bovis and M. tuberculosis strains, respectively. The accuracy of the assay, in terms of specificity, was 100%, as none of the NTM strains tested revealed any amplification product. As little as 20 pg of genomic DNA could be detected, justifying the sensitivity of the method. The m-PCR assay is an extremely useful, simple, reliable and rapid method for routine differential identification of cultures of M. bovis and M. tuberculosis. This m-PCR may be a valuable diagnostic tool in areas of endemicity, where bovine and human tuberculosis coexist, and the distinction of M. bovis from M. tuberculosis is required for monitoring the spread of M. bovis to humans.

Animals↗

PubMed2000

Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci.

Conditions have been optimized for the use of a multiplex PCR for the detection of vancomycin-resistant enterococci in nosocomial surveillance specimens. Seven primer sets targeting the genes vanA, vanB, vanC1, vanC2/C3 Enterococcus faecalis-specific, Enterococcus faecium-specific, and rrs (16S rRNA) were used in one reaction tube. The PCR method developed in the present study is simple and reliable for the rapid characterization of vancomycin-resistant enterococci.

Cross Infection↗

PubMed2005

Single multiplex PCR assay to identify simultaneously the six categories of diarrheagenic Escherichia coli associated with enteric infections.

We designed a multiplex PCR for the detection of all categories of diarrheagenic Escherichia coli. This method proved to be specific and rapid in detecting virulence genes from Shiga toxin-producing (stx(1), stx(2), and eae), enteropathogenic (eae and bfp), enterotoxigenic (st II and lt), enteroinvasive (vir F and ipa H), entero-aggregative (aaf II), and diffuse adherent (daa E) Escherichia coli in stool samples.

Bacterial Toxins↗

PubMed2003

A novel multiplex PCR system consisting of Y-STRs DYS441, DYS442, DYS443, DYS444, and DYS445.

We have developed a new sensitive multiplex PCR system consisting of five male-specific and polymorphic tetranucleotide STRs--DYS441 (GDB: 10013873), DYS442 (GDB: 10030304), DYS443 (GDB: 10807127), DYS444 (GDB: 10807128), and DYS445 (GDB: 10807129) on the Y chromosome. Fifty pg DNA per 10 microL reaction volume was required for the correct typing of five STRs. Using this system, the five Y-STRs were correctly typed from blood and semen stains that had been stored for several years at room temperature.

Blood Stains↗

PubMed2000

Portuguese population and paternity investigation studies with a multiplex PCR--the AmpFlSTR Profiler Plus.

A Portuguese Caucasian population of 146 unrelated individuals was studied. DNA samples were amplified by multiplex PCR for D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 using the AmpFlSTR Profiler Plus PCR Amplification Kit (Perkin-Elmer). All loci met Hardy-Weinberg expectations. Forensic statistical parameters were according to those obtained by other authors. Statistical differences were observed concerning three loci when comparing the Portuguese Caucasian population and an Italian Caucasian population, although these differences mainly concern the less frequent alleles. Eighty-three paternity investigation cases were analysed. Exclusions in between three and nine loci were observed in all the 23 exclusion cases obtained. Most of the non-exclusion cases had probability of paternity > 99.9%. Two cases with an isolated genetic incompatibility between the alleged father and the child were detected, which may indicate probable mutation cases. These results demonstrate that the AmpFlSTR Profiler Plus is a suitable multiplex for paternity investigation in the Portuguese population.

Alleles↗

PubMed1996

Hungarian population data on six STR loci--HUMVWFA31, HUMTH01, HUMCSF1PO, HUMFES/FPS, HUMTPOX, and HUMHPRTB--derived using multiplex PCR amplification and manual typing.

We present a Hungarian population study for six tetrameric short tandem repeat (STR) loci employing multiplex PCR amplification, electrophoresis of the PCR products in DNA sequencing gels and subsequent detection of allelic fragments by silver staining. The loci were HUMVWFA31, HUMTH01, HUMCSF1PO, HUMFES/ FPS, HUMTPOX, and HUMHPRTB. All loci met Hardy-Weinberg expectations in the examined Hungarian Caucasian population sample (N = 223 individuals). In addition, there was no evidence for association of alleles among the five autosomal loci HUMVWFA31, HUMTH01, HUMCSF1PO, HUMFES/FPS, and HUMTPOX.

Alleles↗

PubMed2005

Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines.

This study describes a simple and rapid multiplex-PCR method to determine the ability to produce histamine, tyramine and putrescine by bacteria. The assay is an improved method based on an assay designed for lactic acid bacteria. This improved method includes a pair of primers based on sequences from histidine decarboxylases from Gram-negative bacteria. Under the optimised conditions, the assay yielded a 367-bp DNA fragment from histidine decarboxylases of Gram-positive bacteria, 534-bp fragment from histidine decarboxylases of Gram-negative bacteria, 924-bp from bacterial tyrosine decarboxylases, and 1446-bp fragment from bacterial ornithine decarboxylases. The method was successfully applied to several biogenic amine-producing bacterial strains, even when DNAs of several target organisms were included in the same reaction. This simple method could be easily incorporated in food control laboratories to detect potentially biogenic amine-producing bacteria in foods.

Bacteriological Techniques↗

PubMed2001

Simultaneous amplification of DNA and RNA virus using multiplex PCR system.

A large number of disease-causing bacteria and viruses are being sequenced and PCR is increasingly used for the diagnosis of the diseases. We have designed a multiplex PCR system for hepatitis B virus (HBV), a DNA virus, and hepatitis E virus (HEV), an RNA virus. A modified technique has been standardized for simultaneous extraction of DNA and RNA, followed by a one-step RT-PCR/PCR.

DNA↗

PubMed2002

Rapid automated simultaneous screening of (G1691A) Factor V, (G20210A) prothrombin, and (C677T) methylenetetrahydrofolate reductase variants by multiplex PCR using fluorescence scanning technology.

The Factor V Leiden mutation (G1691A), and mutations in the prothrombin (G20210A) and 5,10-methylenetetrahydrofolate reductase (C677T) genes are common hereditary risk factors associated with venous thrombosis. The aim of this study was to develop an automated, PCR-based genotyping assay for rapid simultaneous screening of these three mutations. We adapted multiplex PCR, using primer modifications to introduce cleavage sites for restriction endonucleases into the fragments bearing each of the mutations. The three mutations were analyzed in a single tube by fluorescence scanning. An internal digestion control was introduced to prevent false-negative results due to incomplete digestion or a total lack of digestion. DNA fragment analysis was carried out using an automated capillary electrophoresis instrument (ABI310). This reliable, efficient, easy-to-use assay can be applied to specimens from large clinical trials and epidemiological surveys.

Factor V↗

PubMed2004

Determination of Cryptococcus neoformans var. neoformans mating type by multiplex PCR.

Mating type plays an important role in the epidemiology and virulence of Cryptococcus neoformans. The present study designed a multiplex PCR method to distinguish the six mating type patterns (Aa, Da, Aalpha, Dalpha, Aa/Dalpha, and Aalpha/Da) of C. neoformans var. neoformans. PCR amplification identified one fragment for Aa (860 bp), Dalpha (413 bp) and Da (645 bp) strains, two fragments for Aalpha (320 and 400 bp) and Aa/Dalpha (860 and 413 bp) strains, and three fragments (645, 400, 320 bp) for an Aalpha/Da strain. The method appears to be a valid, simple and relatively inexpensive tool for epidemiological and virulence studies.

Cryptococcus neoformans↗

PubMed2001

Multiplex PCR for simultaneous identification of Staphylococcus aureus and detection of methicillin and mupirocin resistance.

In this work, we describe a multiplex PCR assay for the detection of clinically relevant antibiotic resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. Conditions were optimized for the simultaneous detection of the 310-, 456-, and 651-bp regions of the mecA (encoding high-level methicillin resistance), ileS-2 (encoding high-level mupirocin resistance), and femB (encoding a factor essential for methicillin resistance) genes, respectively, from a single colony in a single reaction tube. The femB PCR fragment allows the specific identification of S. aureus. Validation of the method was performed using 50 human isolates of methicillin-resistant S. aureus (MRSA) and the appropriate control strains. This assay offers a rapid, simple, feasible, specific, sensitive, and accurate identification of mupirocin-resistant MRSA clinical isolates and could be systematically applied as a diagnostic test in clinical microbiology laboratories, facilitating the design and use of antibiotic therapy.

Anti-Bacterial Agents↗

PubMed2003

Use of a rapid, single-round, multiplex PCR to detect malarial parasites and identify the species present.

A new, rapid assay, based on a single-round, multiplex PCR, can be used to detect Plasmodium falciparum, P. vivax, P. malariae or P. ovale in human blood. The PCR, which targets the conserved 18S small-subunit RNA genes of the parasites, not only permits a malarial infection to be detected but also allows each Plasmodium species present to be identified, even in cases of mixed infection.

Animals↗